Disintegrin homologs

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for zdint1, a novel member of the Disintegrin Proteases. The polypeptides, and polynucleotides encoding them, are believed to be cell-cell interaction modulating and may be used for delivery and therapeutics. The present invention also includes antibodies to the zdint1 polypeptides.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to Provisional Application No.60/092,371 filed on Jul. 10, 1998. Under 35 U.S.C. § 119(e)(1), thisapplication claims benefit of said Provisional Application.

BACKGROUND OF THE INVENTION

[0002] Disintegrins have been shown to bind cell surface molecules,including integrins, on the surface of various cells, such as platelets,fibroblasts, tumor, endothelial, muscle, neuronal, bone, and spermcells. Disintegrins are unique and potentially useful tools forinvestigating cell-matrix and cell-cell interactions. Additionally, theyhave been useful in the development of antithrombotic and antimetastaticagents due to their anti-adhesive, anti-migration of certain tumorcells, and antiangiogenesis activities.

[0003] Families of proteins which have disintegrin domains include ADAMs(A Metalloprotease and Disintegrin), MDCs(Metalloprotease/Disintegrin/Cysteine-rich) and SVMPs (Snake VenomMetalloprotease).

[0004] For a review of ADAMs, see Wolfsberg and White, DevelopmentalBiology, 180:389-401, 1996. ADAMs have been shown to exist asindependent functional units or in conjunction with other members ofthis family in heterodimeric complexes. Some members of the family existin multiple isoforms which may have resulted from alternative splicing.ADAMs proteins have been shown to have adhesive as well as anti-adhesivefunctions. Some members of the ADAMs family have very specific tissuedistribution while others are widely distributed. Not all members ofthis family are capable of manifesting all of the potential functionsrepresented by the domains common to their genetic structure.

[0005] The ADAMs are characterized by having a propeptide domain, ametalloprotease-like domain, a disintegrin-like domain, a cysteine-richdomain, an EGF-like domain, and a cytoplasmic domain.

[0006] A prototypical example of this family is ADAM 12. ADAM 12, alsoknown as meltrin α, has a truncated isoform, as well as a full-lengthisoform, and is involved in muscle cell fusion and differentiation(Gilpin et al., J. Biol. Chem. 273:157-166, 1998).

[0007] Another prototypical example of this family is ADAM 1, whichforms a heterodimer with ADAM 2 and is involved in sperm/egg fusion(Wolfsberg and White, supra).

[0008] The SVMP family is represented by three classes (P-I, P-II, andP-III). All three classes contain propeptide and metalloproteasedomains. The P-II and P-III classes also contain a disintegrin domain,and the P-III class further contains a cysteine-rich domain. Thesedomains are similar in sequence to those found in the ADAMs. Somemembers of the SVMP family have a conserved “RGD” amino acid sequence.This tripeptide has been shown to form a hairpin loop whose conformationcan disrupt the binding of fibrinogen to activated platelets. This RGDsequence may be substituted by RSE, MVD, MSE, and KGD in P-II SVMPs, andby MSEC, RSEC, IDDC, and RDDC (a tripeptide along with acarboxy-terminal cysteine residue) in P-III SVMPs. Thus, these sequencesmay be responsible for integrin binding in the P-II and P-III SVMPs.

[0009] A prototypical example of a SVMP is jararhagin, which mediatesplatelet aggregation by binding to the platelet α2 subunit (GPIa) viathe disintegrin domain followed by proteolysis of the β₁ subunit (GPIIA)(Huang and Liu, J. Toxicol-Toxin Reviews 16: 135-161, 1997).

[0010] The proteins of the Metalloprotease/Disintegrin/Cysteine-richfamily are involved in diverse tasks, ranging from roles infertilization and muscle fusion, TNFα release from plasma membranes,intracellular protein cleavage, and essential functions in neuronaldevelopment (Blobel, Cell 90:589-592, 1997). This family is alsocharacterized by the metalloprotease, disintegrin and cysteine-richdomains, as described above.

[0011] The present invention provides a novel disintegrin homolog andrelated compositions whose uses should be apparent to those skilled inthe art from the teachings herein.

SUMMARY OF THE INVENTION

[0012] Within one aspect, the present invention provides an isolatedpolypeptide molecule comprising a contiguous sequence of 14 amino acidsof SEQ ID NO:2. Within an embodiment the polypeptide molecule comprisesresidues 437 to 450 of SEQ ID NO:2. Within another embodiment, thepolypeptide molecule is between 82 and 232 amino acids in length. Withinfurther embodiments polypeptide molecule is residues 164 to 382 of SEQID NO:2; residues 383 to 464 of SEQ ID NO:2; and/or residues 465 to 696of SEQ ID NO:2.

[0013] Within another aspect, the invention provides an isolatedpolypeptide molecule selected from the group consisting of: a) apolypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; b) apolypeptide molecule comprising residues 383 to 464 of SEQ ID NO:2; c) apolypeptide molecule comprising residues 465 to 696 of SEQ ID NO:2; d) apolypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; e) apolypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2; f) apolypeptide molecule comprising residues 164 to 696 of SEQ ID NO:2; g) apolypeptide molecule comprising residues 383 to 696 of SEQ ID NO:2; h) apolypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2; i) apolypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2; andj) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO:2.

[0014] Within another aspect is provided an isolated polynucleotidemolecule encoding a polypeptide molecule, wherein the polypeptidemolecule comprises a contiguous sequence of 14 amino acids of SEQ IDNO:2. Within an embodiment, the polypeptide molecule comprises residues437 to 450 of SEQ ID NO:2. Within a further embodiment, the polypeptidemolecule is between 82 and 232 amino acids in length. Within furtherembodiments, the polypeptide molecule is residues 164 to 382 of SEQ IDNO:2; residues 383 to 464 of SEQ ID NO:2; and/or residues 465 to-696 ofSEQ ID NO:2.

[0015] Within another aspect, the invention provides an isolatedpolynucleotide molecule encoding a polypeptide molecule, wherein thepolypeptide molecule is selected from the group consisting of: a) apolypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; b) apolypeptide molecule comprising residues 383 to 464 of SEQ ID NO:2; c) apolypeptide molecule comprising residues 465 to 696 of SEQ ID NO:2; d) apolypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; e) apolypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2; f) apolypeptide molecule comprising residues 164 to 696 of SEQ ID NO:2; g) apolypeptide molecule comprising residues 383 to 696 of SEQ ID NO:2; h) apolypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2; i) apolypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2; andj) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO:2.

[0016] Within another aspect is provided an isolated polynucleotideencoding a fusion protein having a first segment and a second segment,wherein the first segment comprises a first polypeptide encoding apolypeptide having a protease domain and the second segment comprises asecond polynucleotide encoding a polypeptide that has a contiguoussequence of 14 amino acids between residues 383 and 464 of SEQ ID NO:2,and wherein the first segment is positioned amino-terminally to thesecond segment. Within an embodiment, the protease domain is selectedfrom the group consisting of; a) a protease domain that is a member ofthe Disintegrin Proteases; and b) a protease domain that is at least 80%identical to amino acid residues 164 to 382 of SEQ ID NO:2.

[0017] Within another aspect the invention provides an isolatedpolynucleotide molecule encoding a polypeptide molecule wherein thepolynucleotide molecule is selected from the group consisting of: a) apolynucleotide molecule that encodes a polypeptide molecule that is atleast 80% identical to residues 383 to 464 of SEQ ID NO:2; and b) apolynucleotide molecule that is complementary to a). Within anembodiment, the polynucleotide molecule is selected from the groupconsisting of: a) a polynucleotide molecule that encodes a polypeptidemolecule that is at least 80% identical to residues 383 to 696 of SEQ IDNO:2; and b) a polynucleotide molecule that is complementary to a).Within a further embodiment, the polynucleotide molecule is selectedfrom the group consisting of: a) a polynucleotide molecule that encodesa polypeptide molecule that is at least 80% identical to residues 1 to696 of SEQ ID NO:2; and b) a polynucleotide molecule that iscomplementary to a).

[0018] Within another aspect is provided an expression vector comprisingthe following operably linked elements: a) a transcription promoter; b)a DNA segment encoding the polypeptide of claim 1; and c) atranscription terminator. Within an embodiment the DNA segment furtherencodes an affinity tag.

[0019] Within another aspect, the invention provides a cultured cellinto which has been introduced said expression vector, wherein the cellexpresses the polypeptide encoded by the DNA segment.

[0020] Within another aspect, the invention provides a method ofproducing a polypeptide comprising culturing the cell expressing thepolypeptide encoded by the DNA segment; and recovering the polypeptide.

[0021] Within another aspect is provided a method for modulatingcell-cell interactions by combining the polypeptide comprising thesequence of 14 contiguous amino acids, with cells in vivo and in vitro.Within an embodiment, the cells are derived from tissues selected fromthe group consisting of: a) tissues from heart; b) tissues from brain;c) tissues from spinal cord; and d) tissues from skeletal muscle.

[0022] Within another aspect, the invention provides an isolatedpolypeptide molecule comprising a contiguous sequence of amino acids,wherein the contiguous sequence of amino acids is selected from thegroup consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8; c) SEQ ID NO:9; d)SEQ ID NO:10; and e) SEQ ID NO:11.

[0023] Within another aspect is provide an isolated polynucleotidemolecule encoding an isolated polypeptide molecule, wherein thepolypeptide comprises a contiguous sequence of amino acids and isselected from the group consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8;c) SEQ ID NO:9; d) SEQ ID NO:10; and e) SEQ ID NO:11.

[0024] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a Hopp/Woods hydrophilicity profile of the zdint1protein sequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the figure bylower case letters.

[0026]FIG. 2 schematically shows a domain level alignment of members ofADAMs, MDCs, and SVMPs. DISA_TRIGA is a SVMP. MS2_HUMAN is an ADAM.HSUTSP1 (TACE) is a MDC. And HSU52370_(—)1 is fertilin-β, ADAM 2. “sig”denotes the secretory signal peptide; “propep” denotes the propeptidedomain; “Metal-protease” denotes the metalloprotease domain; “disint”denotes the disintegrin domain; “cys” denotes the cysteine-rich domain;“RGD” denotes a tripeptide, Arginine-Glycine-Asparagine; and “TMD”denotes the transmembrane domain.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0028] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0029] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0030] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0031] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0032] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0033] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGAGCTT-3′ are5′-AGCTTgagt-3′ and 3′-tcgacTACC-5′.

[0034] The term “corresponding to”, when applied to positions of aminoacid residues in sequences, means corresponding positions in a pluralityof sequences when the sequences are optimally aligned.

[0035] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0036] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0037] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0038] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0039] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0040] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0041] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, a-globin, b-globin, and myoglobin are paralogs of eachother.

[0042] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules, it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus, all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

[0043] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0044] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0045] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0046] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain or multi-peptide structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. Binding of ligand to receptorresults in a conformational change in the receptor that causes aninteraction between the effector domain and other molecule(s) in thecell. This interaction in turn leads to an alteration in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, increases in cyclic AMP production, mobilization ofcellular calcium, mobilization of membrane lipids, cell adhesion,hydrolysis of inositol lipids and hydrolysis of phospholipids. Ingeneral, receptors can be membrane bound, cytosolic or nuclear;monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergicreceptor) or multimeric (e.g., PDGF receptor, growth hormone receptor,IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptorand IL-6 receptor).

[0047] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0048] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0049] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0050] All references cited herein are incorporated by reference intheir entirety.

[0051] The present invention is based upon the discovery of a novel cDNAsequence (SEQ ID NO:1) and corresponding polypeptide (SEQ ID NO:2)having homology to disintegrin-like family members (ADAMs, SVMPs andMDCs; referred to herein as Disintegrin Proteases, or “DPs”). See, forexample, Blobel, Cell 90:589-592, 1997, and Wolfsberg and White,Developmental Biology 180:389-401, 1996. Disintegrins can be involvedin, for example, anticoagulation, fertilization, muscle fusion,connective tissue disorders, chondrogenesis, arthritis, metastasis andneurogenesis.

[0052] The secretory signal (also known as a leader sequence, preprosequence or pre sequence) domain of these polypeptides directs thepolypeptide through a secretory pathway of a cell in which it issynthesized. The secretory signal and propeptide domain are cleaved fromthe full length molecule, resulting in the mature form of the zdint1polypeptide. The protease domain may be active or inactive. Some membersof the disintegrin family have “active” zinc catalytic sites which maybe regulated by a “cysteine-switch” in the cysteine-rich domain.Examples of family members which have “active” protease domains are ADAM1 and ADAM 10, which are involved in sperm/egg fusion and degradation ofmyelin basic sheath protein, respectively. Other members of this familydo not have such a catalytic site and are “inactive”. An example of afamily member which contains an inactive protease domain is ADAM 11,which may be involved in tumor suppression. Other protein families whichare known to have inactive protease domains are the serine proteases.

[0053] The adhesion (disintegrin) domain of this protein binds integrindomains on the surface a multitude of cells, depending on thespecificity of the disintegrin. The predicted binding site within thisdisintegrin domain is often an amino acid loop comprising about 13 aminoacids. The conformation of this sequence upon folding results in ahairpin loop presenting an amino acid sequence at its tip. This sequenceis often “RGD”, but may be substituted by a variety of other amino acidresidues (Wolfsberg and White, supra; and Jia, J. Biol. Chem.272:13094-13102 1997). The diversity of these sequences may reflectthat: 1) not all disintegrin domains serve as ligands for integrins (orother cell surface receptors); 2) disintegrin domains with differentsequences bind to different types of cell surface receptors; or 3) theimportant part of the disintegrin structure loop is its structure, notits sequence, and thus, that the receptors for the specific classes ofdisintegrin domains can recognize a multitude of disintegrin bindingloop sequences. Disintegrin domains have been shown to be responsiblefor cell-cell interactions, including inhibition of platelet aggregationby binding GPIIb/IIIa (fibronectin receptor) and/or GPIa/IIa (collagenreceptor) as well as cell fusion.

[0054] Many disintegrin family members have a fusion domain, arelatively hydrophobic domain of about 23 amino acids. This domain ispresent within some of the ADAM family members, and has been shown to beinvolved in cell-cell fusion, and particularly in sperm/egg fusion, andmuscle fusion.

[0055] The cysteine-rich domain varies in the DP family members and isbelieved to be involved in structurally presenting the integrin-bindingregion to integrins.

[0056] Many DP family members have a transmembrane domain, which acts toanchor the polypeptide to the cell membrane.

[0057] The signaling domain of disintegrin family members tends to beconserved in length and sites for phosphorylation. However, beyond thatthey tend to be unique in amino acid composition. Some disintegrinfamily members may signal by binding to the SH3 domain of Abl, Src,and/or Src-related SH3 domains.

[0058] The zdint1 polypeptides of the present invention are a novelmember of the DP family. The presence of isoforms of zdint1 which alsocomprise a transmembrane domain would suggest that zdint1 will have analternatively spliced variant with a signaling domain.

[0059] The novel zdint1 polypeptide-encoding polynucleotides of thepresent invention were initially identified by performing a Blastsimilarity search. An expressed sequence tag corresponding tonucleotides 1097 to 1415 of SEQ ID NO:1 was used to obtain a clone thathad been isolated from an infant brain plasmid library.

[0060] Examination of the zdint1 deduced amino acid sequence (SEQ IDNO:2) permitted identification of the following domains: a propeptidesequence, ending at residue 163 of SEQ ID NO: 2; a protease sequence,residues 164 to 382 of SEQ ID NO: 2; a disintegrin sequence, residues383 to 464 of SEQ ID NO: 2; and a cysteine-rich sequence, residues 465to 696 of SEQ ID NO:2. Within the disintegrin domain, there is a“disintegrin loop” sequence, residues 438 to 449 of SEQ ID NO:2. Theamino acid sequence, ECD, which corresponds to residues 443 to 445 ofSEQ ID NO: 2, is analogous to the “RGD binding loop” of some othermembers of the DPs.

[0061] Analysis of tissue distribution of zdint1 was performed by theNorthern blotting technique using Human Multiple Tissue, Master Dot, andhuman vascular blots. Strong signals of three transcript sizes,approximately 3.0 kb, 4.4 kb, and 7.5 kb, were observed in heart on themultiple tissue Northern blots. Faint signals of the same transcriptsizes were observed in brain and spinal cord. Fainter signals of thethree transcript sizes were observed in skeletal muscle. The Master DotBlot showed strong signals in brain, heart, fetal brain, and fetalheart. The human vascular blot showed a strong signal at 3-3.5 kb inhuman aortic endothelial cells and weaker signals in aortic smoothmuscle cells and normal human lung fibroblast cells.

[0062] The protease domain of zdint1 has 49.5% identity to the proteasedomain of the nearest family neighbor, ADAM 11, at the polypeptidelevel, and 58% identity at the polynucleotide level. The disintegrindomain of zdint1 has 66.7% identity to the disintegrin domain of thenearest family neighbor, ADAM 11, at the polypeptide level, and 64.3%identity at the polynucleotide level. The expression of ADAM 11 has beenshown to decrease in breast cancer tissues and, thus, is suggested toact as a tumor suppresser in breast cancer (Emi et al., Nature Gen.5:151-157, 1993). Additionally, ADAM 11 has been shown to have multipleisoforms as a result of alternative splicing.

[0063] Another protein which is an example of alternative splicing inthe DPs is ADAM 12, meltrin α. The truncated form of this molecule,which lacks the propeptide and metalloprotease domains, is associatedwith ectopic muscle formation in vivo, but not in vitro, indicating thatcells expressing this gene produce a growth factor that acts onneighboring progenitor cells.

[0064] Other ADAMs have been considered for treating angioplasty, acutecoronary syndrome, prevention of restenosis on stents, and prevention ofexcess adhesion following surgical procedures, prevention of metastasis,as well as for degradation of specific proteins, such as, for example,amyloid precursor protein.

[0065] Polynucleotides:

[0066] The highly conserved amino acids in the disintegrin domain ofzdint1 can be used as a tool to identify new family members. Forinstance, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding the conserved disintegrin domainfrom RNA obtained from a variety of tissue sources or cell lines. Inparticular, highly degenerate primers designed from the zdint1 sequencesare useful for this purpose.

[0067] The present invention also provides polynucleotide molecules,including DNA and RNA molecules, that encode the zdint1 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate DNA sequence that encompasses all DNAs that encode thezdint1 polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:3 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, zdint1polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 2088 of SEQ ID NO:3 and their RNA equivalents arecontemplated by the present invention. Table 1 sets forth the one-lettercodes used within SEQ ID NO:3 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C. TABLE 1Nucleotide Resolution Nucleotidie Complement A A T T C C G G G G C C T TA A R A | G Y C | T Y C | T R A | G M A | C K G | T K G | T M A | C S C| G S C | G W A | T W A | T H A | C | T D A | G | T B C | G | T V A | C| G V A | C | G B C | G | T B A | G | T H A | C | T N A | C | G | T N A| C | G | T

[0068] The degenerate codons used in SEQ ID NO:3, encompassing allpossible codons for a given amino acid, are set forth in Table 2. TABLE2 One Degen- Amino Letter erate Acid Code Codons Codon Cys C TGC TGT TGYSer S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCACCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN AsnN AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR HisH CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn | Asp B RAY Glu | Gln Z SAR Any X NNN

[0069] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0070] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0071] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typical stringent conditions are those in whichthe salt concentration is up to about 0.03 M at pH 7 and the temperatureis at least about 60° C.

[0072] The isolated polynucleotides of the present invention include DNAand RNA. Methods for preparing DNA and RNA are well known in the art. Ingeneral, RNA is isolated from a tissue or cell that produces largeamounts of zdint1 RNA. Such tissues and cells are identified by Northernblotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and includeheart, brain, skeletal muscle, spinal cord, fetal heart, and fetalbrain. Total RNA can be prepared using guanidine HCl extraction followedby isolation by centrifugation in a CsCl gradient (Chirgwin et al.,Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNAusing the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. In the alternative, genomic DNA can beisolated. Polynucleotides encoding zdint1 polypeptides are thenidentified and isolated by, for example, hybridization or PCR.

[0073] A full-length clone encoding zdint1 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to zdint1 or other specificbinding partners.

[0074] Zdint1 polynucleotide sequences disclosed herein can also be usedas probes or primers to clone 5′ non-coding regions of a zdint1 gene. Inview of the tissue-specific expression observed for zdint1 by Northernblotting, this gene region is expected to provide for heart-, brain-,spinal cord-, and skeletal muscle-specific expression. Promoter elementsfrom a zdint1 gene could thus be used to direct the tissue-specificexpression of heterologous genes in, for example, transgenic animals orpatients treated with gene therapy. Cloning of 5′ flanking sequencesalso facilitates production of zdint1 proteins by “gene activation” asdisclosed in U.S. Pat. No. 5,641,670. Briefly, expression of anendogenous zdint1 gene in a cell is altered by introducing into thezdint1 locus a DNA construct comprising at least a targeting sequence, aregulatory sequence, an exon, and an unpaired splice donor site. Thetargeting sequence is a zdint1 5′ non-coding sequence that permitshomologous recombination of the construct with the endogenous zdint1locus, whereby the sequences within the construct become operably linkedwith the endogenous zdint1 coding sequence. In this way, an endogenouszdint1 promoter can be replaced or supplemented with other regulatorysequences to provide enhanced, tissue-specific, or otherwise regulatedexpression.

[0075] The polynucleotides of the present invention can also besynthesized using DNA synthesizers. Currently the method of choice isthe phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. See Glick and Pasternak, MolecularBiotechnology, Principles and Applications of Recombinant DNA, (ASMPress, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53:323-356 (1984) and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637(1990).

[0076] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are zdint1 polypeptides from other mammalianspecies, including murine, porcine, ovine, bovine, canine, feline,equine, and other primate polypeptides. Orthologs of human zdint1 can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses zdint1 as disclosed herein. Such tissue or cell typewould include, for example, heart, brain, spinal cord, and skeletalmuscle. Suitable sources of mRNA can be identified by probing Northernblots with probes designed from the sequences disclosed herein. Alibrary is then prepared from mRNA of a positive tissue or cell line. Azdint1-encoding cDNA can then be isolated by a variety of methods, suchas by probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.Pat. No. 4,683,202), using primers designed from the representativehuman zdint1 sequence disclosed herein. Within an additional method, thecDNA library can be used to transform or transfect host cells, andexpression of the cDNA of interest can be detected with an antibody tozdint1 polypeptide. Similar techniques can also be applied to theisolation of genomic clones.

[0077] Those skilled in the art will recognize that the sequencedisclosed in SEQ ID NO:1 represents a single allele of human zdint1 andthat allelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the zdint1 polypeptide are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

[0078] The present invention also provides isolated zdint1 polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO:2 andtheir orthologs. The term “substantially homologous” is used herein todenote polypeptides having about 50%, preferably 60% more preferably atleast 70%, and even more preferably 80% sequence identity to thesequences shown in SEQ ID NO:2 or their orthologs. Such polypeptideswill more preferably be at least 90% identical, and most preferably 95%or more identical to SEQ ID NO:2 or its orthologs.) Percent sequenceidentity is determined by conventional methods. See, for example,Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 1992. Briefly, twoamino acid sequences are aligned to optimize the alignment scores usinga gap opening penalty of 10, a gap extension penalty of 1, and the“blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown inTable 3 (amino acids are indicated by the standard one-letter codes) Thepercent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0079] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0080] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant zdint1. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

[0081] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

[0082] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with otherparameters set as default.

[0083] The present invention includes nucleic acid molecules that encodea polypeptide having one or more conservative amino acid changes,compared with the amino acid sequence of SEQ ID NO:2. The BLOSUM62 tableis an amino acid substitution matrix derived from about 2,000 localmultiple alignments of protein sequence segments, representing highlyconserved regions of more than 500 groups of related proteins (Henikoffand Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly,the BLOSUM62 substitution frequencies can be used to define conservativeamino acid substitutions that may be introduced into the amino acidsequences of the present invention. As used herein, the language“conservative amino acid substitution” refers to a substitutionrepresented by a BLOSUM62 value of greater than −1. For example, anamino acid substitution is conservative if the substitution ischaracterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

[0084] Variant zdint1 polypeptides or substantially homologous zdint1polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from 383 to 464 amino acid residues thatcomprise a sequence that is at least 50%, preferably at least 60%, andmore preferably 80% or more identical to the corresponding region of SEQID NO:2. Polypeptides comprising affinity tags can further comprise aproteolytic cleavage site between the zdint1 polypeptide and theaffinity tag. Preferred such sites include thrombin cleavage sites andfactor Xa cleavage sites. TABLE 4 Conservative amino acid substitutionsBasic: arginine lysine histidine Acidic: glutamic acid aspartic acidPolar: glutamine asparagine Hydrophobic: leucine isoleucine valineAromatic: phenylalanine tryptophan tyrosine Small: glycine alanineserine threonine methionine

[0085] The present invention further provides a variety of polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a disintegrin polypeptide domain canbe prepared as a fusion to a dimerizing protein, as disclosed in U.S.Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include other disintegrin polypeptide domains or disintegrinpolypeptide domain fragments. Disintegrin polypeptide domain fusions, ordisintegrin polypeptide domain fragment fusions, can be expressed ingenetically engineered cells to produce a variety of multimericdisintegrin-like analogs. Auxiliary domain polypeptides can be fused todisintegrin domain polypeptides to target them to specific cells,tissues, or macromolecules (e.g., heart, brain, spinal cord, skeletalmuscle, platelets). For example, a protease polypeptide domain, orprotease polypeptide fragment or protein, could be targeted to apredetermined cell type by fusing it to a disintegrin polypeptide domainor fragment that specifically binds to an integrin polypeptide orintegrin-like polypeptide on the surface of the target cell. In thisway, polypeptides, polypeptide fragments and proteins can be targetedfor therapeutic or diagnostic purposes. Such disintegrins or proteasepolypeptide domains or fragments can be fused to two or more moieties,such as an affinity tag for purification and a targeting-disintegrindomain. Polypeptide fusions can also comprise one or more cleavagesites, particularly between domains. See Tuan et al., Connective TissueResearch 34:1-9, 1996.

[0086] Polypeptide fusions of the present invention will generallycontain not more than about 1,500 amino acid residues, preferably notmore than about 1,200 residues, more preferably not more than about1,000 residues, and will in many cases be considerably smaller. Forexample, residues of zdint1 polypeptide can be fused to E. coliβ-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol.143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xacleavage site. In a second example, residues of zdint1 polypeptide canbe fused to maltose binding protein (approximately 370 residues), a4-residue cleavage site, and a 6-residue polyhistidine tag.

[0087] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0088] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zdint1 aminoacid residues.

[0089] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity asdisclosed below to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699-708, 1996. Sites of disintegrin-integrin, or proteaseinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol.Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.The identities of essential amino acids can also be inferred fromanalysis of homologies with related disintegrin-like molecules.

[0090] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

[0091] Variants of the disclosed zdint1 DNA and polypeptide sequencescan be generated through DNA shuffling, as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the-desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0092] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., disintegrin-cellsurface binding or protease activity) can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

[0093] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptide fragments orvariants of SEQ ID NO:2 or that retain the disintegrin and or proteaseactivity of the wild-type zdint1 protein. Such polypeptides may includeadditional amino acids from, for example, a secretory domain, apropeptide domain, a protease domain, part or all of a transmembrane andintracellular domains, including amino acids responsible forintracellular signaling; a fusion domains; affinity tags; and the like.

[0094] For any zdint1 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above.

[0095] Protein Production

[0096] The zdint1 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

[0097] In general, a DNA sequence encoding a zdint1 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0098] To direct a zdint1 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be derived from another secretedprotein (e.g., t-PA) or synthesized de novo. The secretory signalsequence is operably linked to the zdint1 DNA sequence, i.e., the twosequences are joined in the correct reading frame and positioned todirect the newly synthesized polypeptide into the secretory pathway ofthe host cell. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the polypeptide of interest, although certainsecretory signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830). Polypeptides and peptidefragments of the present invention are considered biologically active inthe absence of the native signal sequence.

[0099] The protease domain of zdint1 can be substituted by aheterologous sequence providing a different protease domain. In thiscase, the fusion product can be secreted, and the disintegrin domain ofzdint1 can direct the protease domain to a specific tissue describedabove. This substituted protease domain can be chosen from the proteasedomains represented by the DP protein families, or domains from otherknown proteases.

[0100] Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0101] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins, such as CD4, CD8, Class I MHC, or placental alkalinephosphatase, may be used to sort transfected cells from untransfectedcells by such means as FACS sorting or magnetic bead separationtechnology.

[0102] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalifornica nuclear polyhedrosis virus (AcNPV). See, King, L. A. andPossee, R. D., The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methodsin Molecular Biology, Totowa, N.J., Humana Press, 1995. A second methodof making recombinant zdint1 baculovirus utilizes a transposon-basedsystem described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,1993). This system, which utilizes transfer vectors, is sold in theBac-to-Bac™ kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, pFastBac1™ (Life Technologies) containing aTn7 transposon to move the DNA encoding the zdint1 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins, M. S. and Possee, R. D., J Gen Virol71:971-6, 1990; Bonning, B. C. et al., J Gen Virol 75:1551-6, 1994; and,Chazenbalk, G. D., and Rapoport, B., J Biol Chem 270:1543-9, 1995. Inaddition, transfer vectors can include an in-frame fusion with DNAencoding an epitope tag at the C- or N-terminus of the expressed zdint1polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. etal., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known inthe art, a transfer vector containing zdint1 is transformed into E.Coli, and screened for bacmids which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is isolated, using common techniques, andused to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses zdint1 is subsequently produced.Recombinant viral stocks are made by methods commonly used the art.

[0103] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. U.S. Pat. No. 5,300,435).Commercially available serum-free media are used to grow and maintainthe cells. Suitable media are Sf900 II™ (Life Technologies) or ESF ₉₂₁™(Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. The cells are grown up from an inoculation density ofapproximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells at which timea recombinant viral stock is added at a multiplicity of infection (MOI)of 0.1 to 10, more typically near 3. Procedures used are generallydescribed in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.).Subsequent purification of the zdint1 polypeptide from the supernatantcan be achieved using methods described herein.

[0104] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0105] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0106] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zdint1polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0107] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0108] Protein Isolation

[0109] It is preferred to purify the polypeptides of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

[0110] Expressed recombinant zdint1 polypeptides (or chimeric zdint1polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

[0111] The polypeptides of the present invention can be isolated by acombination of procedures including, but not limited to, anion andcation exchange chromatography, size exclusion, and affinitychromography. See Example 3 for a procedure. For example, immobilizedmetal ion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

[0112] Fragments/Fusion Proteins

[0113] To direct the export of a zdint1 polypeptide from the host cell,the zdint1 DNA is linked to a second DNA segment encoding a secretorypeptide, such as a t-PA secretory peptide. To facilitate purification ofthe secreted receptor polypeptide, a C-terminal extension, such as apoly-histidine tag, substance P, Flag peptide (Hopp et al.,Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.) or another polypeptide or protein for which an antibody orother specific binding agent is available, can be fused to the zdint1polypeptide.

[0114] Moreover, using methods described in the art, polypeptidefusions, or hybrid zdint1 proteins, are constructed using regions ordomains of the inventive zdint1 in combination with those of otherdisintegrin-like molecules. (e.g. ADAM, MDC, and SVMP), or heterologousproteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur.Opin. Biology, 5:511-5, 1994, and references therein). These methodsallow the determination of the biological importance of larger domainsor regions in a polypeptide of interest. Such hybrids may alter reactionkinetics, binding, constrict or expand the substrate specificity, oralter tissue and cellular localization of a polypeptide, and can beapplied to polypeptides of unknown structure.

[0115] Fusion proteins can be prepared by methods known to those skilledin the art by preparing each component of the fusion protein andchemically conjugating them. Alternatively, a polynucleotide encodingboth components of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a domains conferring a biologicalfunction may be swapped between zdint1 of the present invention with thefunctionally equivalent domains from another family member, such asADAM, MDC, and SVMP. Such domains include, but are not limited to,conserved motifs such as the secretory signal sequence, protease, RGD,cysteine, and disintegrin domains. Such fusion proteins would beexpected to have a biological functional profile that is the same orsimilar to polypeptides of the present invention or other knowndisintegrin-like family proteins (e.g. ADAMs, MDCs, and SVMPs),depending on the fusion constructed. Moreover, such fusion proteins mayexhibit other properties as disclosed herein.

[0116] zdint1 polypeptides or fragments thereof may also be preparedthrough chemical synthesis. zdint1 polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

[0117] Chemical Synthesis of Polypeptides

[0118] Zdint1 polypeptides, peptides, variants and or fragments thereofmay also be prepared through chemical synthesis. TML polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; amidated or non-amidated; sulfated or non-sulfated; andmay or may not include an initial methionine amino acid residue. Forexample, TML polypeptides can also be synthesized by exclusive solidphase synthesis, partial solid phase methods, fragment condensation orclassical solution synthesis. The polypeptides are preferably preparedby solid phase peptide synthesis, for example as described byMerrifield, J. Am. Chem. Soc. 85:2149, 1963. The synthesis is carriedout with amino acids that are protected at the alpha-amino terminus.Trifunctional amino acids with labile side-chains are also protectedwith suitable groups to prevent undesired chemical reactions fromoccurring during the assembly of the polypeptides. The alpha-aminoprotecting group is selectively removed to allow subsequent reaction totake place at the amino-terminus. The conditions for the removal of thealpha-amino protecting group do not remove the side-chain protectinggroups.

[0119] The alpha-amino protecting groups are those known to be useful inthe art of stepwise polypeptide synthesis. Included are acyl typeprotecting groups (e.g., formyl, trifluoroacetyl, acetyl), aryl typeprotecting groups (e.g., biotinyl), aromatic urethane type protectinggroups [e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexloxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tBoc and Fmoc.

[0120] The side-chain protecting groups selected must remain intactduring coupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

[0121] In tBoc chemistry, the preferred side-chain protecting groups aretosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutamine and histidine, tert-butyl foraspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc forlysine and tryptophan.

[0122] For the synthesis of phosphopeptides, either direct orpost-assembly incorporation of the phosphate group is used. In thedirect incorporation strategy, the phosphate group on serine, threonineor tyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphoramidite and then oxidized bytert-butylhydroperoxide.

[0123] Solid phase synthesis is usually carried out from thecarboxyl-terminus by coupling the alpha-amino protected (side-chainprotected) amino acid to a suitable solid support. An ester linkage isformed when the attachment is made to a chloromethyl, chlortrityl orhydroxymethyl resin, and the resulting polypeptide will have a freecarboxyl group at the C-terminus. Alternatively, when an amide resinsuch as benzhydrylamine or p-methylbenzhydrylamine resin (for tBocchemistry) and Rink amide or PAL resin (for Fmoc chemistry) are used, anamide bond is formed and the resulting polypeptide will have acarboxamide group at the C-terminus. These resins, whether polystyrene-or polyamide-based or polyethyleneglycol-grafted, with or without ahandle or linker, with or without the first amino acid attached, arecommercially available, and their preparations have been described byStewart et al., “Solid Phase Peptide Synthesis” (2nd Edition), (PierceChemical Co., Rockford, Ill., 1984) and Bayer & Rapp Chem. Pept. Prot.3:3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: APractical Approach, IRL Press, Oxford, 1989.

[0124] The C-terminal amino acid, protected at the side chain ifnecessary, and at the alpha-amino group, is attached to a hydroxylmethylresin using various activating agents including dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole(CDI). It can be attached to chloromethyl or chlorotrityl resin directlyin its cesium tetramethylammonium salt form or in the presence oftriethylamine (TEA) or diisopropylethylamine (DIEA). First amino acidattachment to an amide resin is the same as amide bond formation duringcoupling reactions.

[0125] Following the attachment to the resin support, the alpha-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300-320 nm or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

[0126] Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH2Cl2 or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970.

[0127] After the entire assembly of the desired 5 peptide, thepeptide-resin is cleaved with a reagent with proper scavengers. The Fmocpeptides are usually cleaved and deprotected by TFA with scavengers(e.g., H20, ethanedithiol, phenol and thioanisole). The tBoc peptidesare usually cleaved and deprotected with liquid HF for 1-2 hours at −5to 0° C., which cleaves the polypeptide from the resin and removes mostof the side-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury (II) acetate and alternatively by iodine,thallium(III)trifluoroacetate or silver tetrafluoroborate whichsimultaneously oxidize cysteine to cystine. Other strong acids used fortBoc peptide cleavage and deprotection include trifluoromethanesulfonicacid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

[0128] The disintegrin loop (residue 438 to residue 449 of SEQ ID NO:2)is of particular interest for use in assays and treatment of disordersof the heart, brain, spinal cord, and skeletal muscle. For thesepurposes the disintegrin loop peptide synthesized includes the terminalcysteine residues and thus, would be from residue 437 to residue 450 ofSEQ ID NO:2. This peptide can be synthesized as a linear peptide or adisulfide linked peptide. Peptides having disulfide bonds betweenresidues can be 438, 444, and 450 are of particular interest. See Jia,L. G., ibid for additional description of peptide synthesis anddisulfide linkage.

[0129] One skilled in the art will recognize that it is useful to designand synthesize new binding peptides using the integrin binding peptidesof zdint1 as a model. Methods for synthesizing such peptides aredescribed by P. L. Barker et al., J. Med. Chem. 35: 2040-2048, 1992, andL. Jia et al., J. Biol. Chem. 272: 13094-13102, 1997. As the structuralconformation of the integrin binding peptide is critical, it isrecognized that although some amino acid substitutions will not changethe conformation of the peptides, the cyclization of the peptide isadvantageously conserved. Synthetic peptides are useful as agonists orantagonsits for zdint1 and could be assayed.

[0130] Assays

[0131] The activity of zdint1 polypeptides can be measured using avariety of assays that measure, for example, cell-cell interactions,proteolysis, extracellular matrix formation or remodeling. Additionally,other biological functions associated with disintegrin family members orwith integrin/disintegrin interactions, apoptosis, proliferation ordifferentiation can also be measured. Of particular interest is a changein platelet aggregation. Assays measuring platelet aggregation are wellknown in the art. For a general reference, see Dennis, Proc. Natl. Acad.Sci. 87: 2471-2475, 1989.

[0132] Another assay of interest measures or detects changes indifferentiation, development and/or and electrical coupling of musclecells or myocytes. Additionally, the effects of a zdint1 polypeptides oncell-cell interactions of fibroblasts, myoblasts, nerve cells, whiteblood cells, endothelial cells and tumor cells would be of interest tomeasure. Yet another assays examines changes in protease activity andapoptosis.

[0133] The activity of molecules of the present invention can bemeasured using a variety of assays that, for example, measure neogenesisor hyperplasia (i.e., proliferation) of cardiac cells based on thetissue specificity in adult heart. Additional activities likelyassociated with the polypeptides of the present invention includeproliferation of endothelial cells, cardiomyocytes, fibroblasts,skeletal myocytes directly or indirectly through other growth factors;action as a chemotaxic factor for endothelial cells, fibroblasts and/orphagocytic cells; osteogenic factor; and factor for expandingmesenchymal stem cell and precursor populations.

[0134] Proliferation can be measured using cultured cardiac cells or invivo by administering molecules of the claimed invention to anappropriate animal model. Generally, proliferative effects are observedas an increase in cell number and therefore, may include inhibition ofapoptosis, as well as mitogenesis. Cultured cells include cardiacfibroblasts, cardiac myocytes, skeletal myocytes, human umbilical veinendothelial cells from primary cultures. Established cell lines include:NIH 3T3 fibroblast (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No.CRL-1680), H9c2 rat heart myoblasts (ATCC No. CRL-1446), Shionogimammary carcinoma cells (Tanaka et al., Proc. Natl. Acad. Sci.89:8928-8932, 1992) and LNCap.FGC adenocarcinoma cells (ATCC No.CRL-1740). Assays measuring cell proliferation are well known in theart. For example, assays measuring proliferation include such assays aschemosensitivity to neutral red dye (Cavanaugh et al., InvestigationalNew Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides(Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use oftetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley etal., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84,1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988).

[0135] Differentiation is a progressive and dynamic process, beginningwith pluripotent stem cells and ending with terminally differentiatedcells. Pluripotent stem cells that can regenerate without commitment toa lineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population. Myocytes, osteoblasts, adipocytes, chrondrocytes,fibroblasts and reticular cells are believed to originate from a commonmesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).Markers for mesenchymal stem cells have not been well identified (Owenet al., J. of Cell Sci. 87:731-738, 1987), so identification is usuallymade at the progenitor and mature cell stages. The existence of earlystage cardiac myocyte progenitor cells (often referred to as cardiacmyocyte stem cells) has been speculated, but not demonstrated, in adultcardiac tissue. The novel polypeptides of the present invention areuseful for studies to isolate mesenchymal stem cells and cardiac myocyteprogenitor cells, both in vivo and ex vivo.

[0136] There is evidence to suggest that factors that stimulate specificcell types down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, zdint1 polypeptides may stimulateinhibition or proliferation of myocytes, smooth muscle cells,osteoblasts, adipocytes, chrondrocytes and endothelial cells. Moleculesof the present invention may, while stimulating proliferation ordifferentiation of cardiac myocytes, inhibit proliferation ordifferentiation of adipocytes, by virtue of their effect on commonprecursor/stem cells. Thus, molecules of the present invention have usein inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.

[0137] Assays measuring differentiation include, for example, measuringcell-surface markers associated with stage-specific expression of atissue, enzymatic activity, functional activity or morphological changes(Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994;Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; allincorporated herein by reference).

[0138] In vivo assays for evaluating cardiac neogenesis or hyperplasiainclude treating neonatal and mature rats with the molecules of thepresent invention. The animals' cardiac function is measured as heartrate, blood pressure, and cardiac output to determine left ventricularfunction. Post-mortem methods for assessing cardiac improvement include:increased cardiac weight, nuclei/cytoplasmic volume, staining of cardiachistology sections to determine proliferating cell nuclear antigen(PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res.75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci.93:8630-8635, 1996.) Assays measuring in vivo effects of syntheticzdint1 agonists include a Left Ventricular Hypertrophy model (A. M.Feldman et al., Circ. Res. 73: 184-192, 1993), which measures remodelingand repair after congestive heart failure and chronic pressure overload.

[0139] Proteins, including alternatively spliced peptides, of thepresent invention are useful for tumor suppression, and growth anddifferentiation either working in isolation, or in conjunction withother molecules (growth factors, cytokines, etc.) in brain, heart,spinal column, and skeletal muscle cells. Alternative splicing of zdint1may be cell-type specific and confer activity to specific tissues.

[0140] Proteins of the present invention are useful for delivery oftherapeutic agents such as, but not limited to, proteases,radionuclides, chemotherapy agents, and small molecules. Effects ofthese therapeutic agents can be measured in vitro using cultured cellsor in vivo by administering molecules of the claimed invention to theappropriate animal model. For instance, zdint1 transfected expressionhost cells may be embedded in an alginate environment and injected(implanted) into recipient animals. Alginate-poly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers have been described as a means to entrap transfected mammaliancells or primary mammalian cells. These types of non-immunogenic“encapsulations” or microenvironments permit the transfer of nutrientsinto the microenvironment, and also permit the diffusion of proteins andother macromolecules secreted or released by the captured cells acrossthe environmental barrier to the recipient animal. Most importantly, thecapsules or microenvironments mask and shield the foreign, embeddedcells from the recipient animal's immune response. Suchmicroenvironments can extend the life of the injected cells from a fewhours or days (naked cells) to several weeks (embedded cells).

[0141] Alginate threads provide a simple and quick means for generatingembedded cells. The materials needed to generate the alginate threadsare readily available and relatively inexpensive. Once made, thealginate threads are relatively strong and durable, both in vitro and,based on data obtained using the threads, in vivo. The alginate threadsare easily manipulable and the methodology is scalable for preparationof numerous threads. In an exemplary procedure, 3% alginate is preparedin sterile H₂O, and sterile filtered. Just prior to preparation ofalginate threads, the alginate solution is again filtered. Anapproximately 50% cell suspension (containing about 5×10⁵ to about 5×10⁷cells/ml) is mixed with the 3% alginate solution. One ml of thealginate/cell suspension is extruded into a 100 mM sterile filteredCaCl₂ solution over a time period of −15 min, forming a “thread”. Theextruded thread is then transferred into a solution of 50 mM CaCl₂, andthen into a solution of 25 mM CaCl₂. The thread is then rinsed withdeionized water before coating the thread by incubating in a 0.01%solution of poly-L-lysine. Finally, the thread is rinsed with LactatedRinger's Solution and drawn from solution into a syringe barrel (withoutneedle attached) A large bore needle is then attached to the syringe,and the thread is intraperitoneally injected into a recipient in aminimal volume of the Lactated Ringer's Solution.

[0142] An alternative in vivo approach for assaying proteins of thepresent invention involves viral delivery systems. Exemplary viruses forthis purpose include adenovirus, herpesvirus, lentivirus, vaccinia virusand adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acid (for a review, see T. C. Becker et al.,Meth. Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel,Science & Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: adenovirus can (i) accommodate relatively large DNA inserts;(ii) be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) be used with a large number of available vectorscontaining different promoters. Also, because adenoviruses are stable inthe bloodstream, they can be administered by intravenous injection.

[0143] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential El gene has been deleted from the viral vector, and the viruswill not replicate unless the E1 gene is provided by the host cell (thehuman 293 cell line is exemplary). When intravenously administered tointact animals, adenovirus primarily targets the liver. If theadenoviral delivery system has host cells. However, the host's tissue(e.g., liver) will express and process (and, if a secretory signalsequence is present, secrete) the heterologous protein. Secretedproteins will enter the circulation in the highly vascularized liver,and effects on the infected animal can be determined.

[0144] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293S cells can be grown in suspension cultureat relatively high cell density to produce significant amounts ofprotein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant. Within the infected 293Scell production protocol, non-secreted proteins may also be effectivelyobtained.

[0145] Within yet another embodiment is provided an oligonucleotideprobe or primer comprising at least 14 contiguous nucleotides of apolynucleotide of SEQ ID NO:1 or a sequence complementary to SEQ IDNO:1.

[0146] Agonists and Antagonists

[0147] In view of the tissue distribution (heart, brain, spinal cord andskeletal muscle) observed for zdint1 expression, agonists (including thenative disintegrin and protease domains) and antagonists have enormouspotential in both in vitro and in vivo applications. Compoundsidentified as zdint1 agonists and antagonists are useful for studyingcell-cell interactions, myogenesis, apoptosis, neurogenesis, connectivetissue disorders, chondrogenesis, arthritis, tumor proliferation andsuppression, extracellular matrix proteins, repair and remodeling ofischemia reperfusion and inflammation in vitro and in vivo. For example,zdint1 and agonist compounds are useful as components of defined cellculture media, and may be used alone or in combination with othercytokines and hormones to replace serum that is commonly used in cellculture. Agonists are thus useful in specifically promoting the growthand/or development of cells of the myeloid lineages in culture.Additionally, zdint1 polypeptides and zdint1 agonists, including smallmolecules are useful as a research reagent, such as for the expansion,differentiation, and/or cell-cell interactions of heart, brain, spinalcord, or skeletal muscle cells. zdint1 polypeptides are added to tissueculture media for these cell types.

[0148] Antagonists

[0149] Antagonists are also useful as research reagents forcharacterizing sites of complementary/anti-complementary interaction.Inhibitors of zdint1 activity (zdint1 antagonists) include anti-zdint1antibodies and soluble zdint1 receptors, as well as other peptidic andnon-peptidic agents (including ribozymes).

[0150] zdint1 can also be used to identify inhibitors (antagonists) ofits activity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of zdint1. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzdint1 activity within a variety of assays designed to measuredisintegrin/integrin binding or the stimulation/inhibition ofzdint1-dependent cellular responses. For example, zdint1-responsive celllines can be transfected with a reporter gene construct that isresponsive to a zdint1-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a DNA response element operably linked to a gene encoding anassayable protein, such as luciferase, or a metabolite. DNA responseelements can include, but are not limited to, cyclic AMP responseelements (CRE), hormone response elements (HRE), insulin responseelement (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7,1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72,1989). Cyclic AMP response elements are reviewed in Roestler et al., J.Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4(8):1087-94; 1990.Hormone response elements are reviewed in Beato, Cell56:335-44; 1989. The most likely reporter gene construct would contain adisintegrin that, upon binding an integrin, would signal intracellularlythrough, for example, a SRE reporter. Candidate compounds, solutions,mixtures or extracts are tested for the ability to inhibit the activityof zdint1 on the target cells, as evidenced by a decrease in zdint1stimulation of reporter gene expression. Assays of this type will detectcompounds that directly block zdint1 binding to cell-surface receptors,i.e., integrin or the anti-complementary member of acomplementary/anti-complementary pair, as well as compounds that blockprocesses in the cellular pathway subsequent tocomplement/anti-complement binding. In the alternative, compounds orother samples can be tested for direct blocking of zdint1 binding to anintegrin using zdint1 tagged with a detectable label (e.g., ¹²⁵I,biotin, horseradish peroxidase, FITC, and the like). Within assays ofthis type, the ability of a test sample to inhibit the binding oflabeled zdint1 to the integrin is indicative of inhibitory activity,which can be confirmed through secondary assays. Integrins used withinbinding assays may be cellular integrins or isolated, immobilizedintegrins.

[0151] An amino acid sequence comprising the “ECD” integrin bindingcomponent of zdint1, (residues 443 to 445 of SEQ ID NO:2), which isanalogous to the “RGD”, integrin binding loop, may also be used as aninhibitor. Such an inhibitor would bind an integrin other than itsnaturally occurring integrin by nature of its folding structure. Aparticular interest in such an inhibitor would be to mediate plateletaggregation. Assays measuring binding and inhibition as well as plateletaggregation are known in the art.

[0152] A zdint1 polypeptide can be expressed as a fusion with animmunoglobulin heavy chain constant region, typically an F_(c) fragment,which contains two constant region domains and lacks the variableregion. Methods for preparing such fusions are disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two non-Ig polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used to evaluate effects andpotential of dimerization of zdint1 with itself or other disintegrinfamily members. Such fusions would also be useful to isolate thecorresponding integrin(s) that zdint1 binds. For use in assays, thechimeras are bound to a support via the Fc region and used in an ELISAformat.

[0153] A zdint1 integrin-binding polypeptide can also be used forpurification of integrin. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingintegrins are passed through the column one or more times to allowintegrins to bind to the integrin binding loop polypeptide. The integrinis then eluted using changes in salt concentration, chaotropic agents(guanidine HCl), or pH to disrupt integrin-receptor binding.

[0154] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complementary/anti-complementary pair) or abinding fragment thereof, and a commercially available biosensorinstrument (BIAcore, Pharmacia Biosensor, Piscataway, N.J.) may beadvantageously employed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulfhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complementary/anti-complementary pair is present in thesample, it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding.

[0155] Another method to assay cell-cell interactions caused by zint1polypeptides, peptides, or variants is with a silicon-based biosensormicrophysiometer which measures the extracellular acidification rate orproton excretion associated with receptor binding and subsequentphysiologic cellular responses. An exemplary device is the Cytosensor™Microphysiometer manufactured by Molecular Devices, Sunnyvale, Calif. Avariety of cellular responses, such as cell proliferation, iontransport, energy production, inflammatory response, regulatory andreceptor activation, and the like, can be measured by this method. See,for example, McConnell, H. M. et al., Science 257:1906-1912, 1992;Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. etal., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zdint1 polypeptide, peptide, variant, agonists, orantagonists. Preferably, the microphysiometer is used to measureresponses of a zdint1-responsive eukaryotic cell, compared to a controleukaryotic cell that does not respond to zdint1 polypeptide, peptide, orvariant. Zdint1-responsive eukaryotic cells comprise cells into which areceptor for zdint1 has been transfected creating a cell that isresponsive to zdint1 polypeptide, peptide, or variant; or cellsnaturally responsive to zdint1 such as, for example, cells derived fromthe kidney or small intestine. Differences, measured by a change, forexample, an increase or diminution in extracellular acidification, inthe response of cells exposed to zdint1 polypeptide, peptide, or variantrelative to a control not exposed to zdint1 polypeptide, peptide, orvariant, are a direct measurement of zdint1-modulated cellularresponses. Moreover, such zdint1-modulated responses can be assayedunder a variety of stimuli. Using the microphysiometer, there isprovided a method of identifying agonists of zdint1 polypeptide,comprising providing cells responsive to a zdint1 polypeptide, culturinga first portion of the cells in the absence of a test compound,culturing a second portion of the cells in the presence of a testcompound, and detecting a change, for example, an increase ordiminution, in a cellular response of the second portion of the cells ascompared to the first portion of the cells. The change in cellularresponse is shown as a measurable change in extracellular acidificationrate. Moreover, culturing a third portion of the cells in the presenceof zdint1 polypeptide and the absence of a test compound can be used asa positive control for the zdint1-responsive cells, and as a control tocompare the agonist activity of a test compound with that of the zdint1polypeptide. Moreover, using the microphysiometer, there is provided amethod of identifying antagonists of zdint1 polypeptide, comprisingproviding cells responsive to a zdint1 polypeptide, culturing a firstportion of the cells in the presence of zdint1 and the absence of a testcompound, culturing a second portion of the cells in the presence ofzdint1 and the presence of a test compound, and detecting a change, forexample, an increase or a diminution in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. The change in cellular response is shown as a measurable changein extracellular acidification rate. Antagonists and agonists, forzdint1 polypeptide, can be rapidly identified using this method.

[0156] Moreover, polypeptides, peptides and variants of zdint1 can beused to identify cells, tissues, or cell lines which respond to azdint1-stimulated pathway. The microphysiometer, described above, can beused to rapidly identify ligand-responsive cells, such as cellsresponsive to zdint1 polypeptides peptides and variants of the presentinvention. Cells can be cultured in the presence or absence of zdint1polypeptides, peptides and variants. Those cells which elicit ameasurable change in extracellular acidification in the presence ofzdint1 polypeptides, peptides and variants are responsive to zdint1.Such cell lines, can be used to identify antagonists and agonists ofzdint1 polypeptide as described above.

[0157] Integrin polypeptides and other receptor polypeptides which binddisintegrin polypeptides, and variants thereof, can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al.,Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0158] A “soluble receptor” is a receptor polypeptide that is not boundto a cell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Receptorpolypeptides are said to be substantially free of transmembrane andintracellular polypeptide segments when they lack sufficient portions ofthese segments to provide membrane anchoring or signal transduction,respectively.

[0159] Soluble forms of zdint1 polypeptides may act as antagonsits tozdint1 polypeptides, and would be useful to modulate the effects ofzdint1 in heart, brain, skeletal muscle and spinal cord. Additionally,soluble zdint1 peptides and fragments can disrupt the integrin-mediatedattachment of a cell to the extracellular matrix.

[0160] Antibodies:

[0161] zdint1 polypeptides can also be used to prepare antibodies thatspecifically bind to zdint1 epitopes, peptides or polypeptides. Thezdint1 polypeptide or a fragment thereof serves as an antigen(immunogen) to inoculate an animal and elicit an immune response.Suitable antigens would include fragments of the zdint1 polypeptideencoded by SEQ ID NO:2 which represent six or more contiguoushydrophilic amino acids. Such antigenic regions would be, for example,from amino acid residue 159 to 164 (SEQ ID NO:7); amino acid residue 158to 163 (SEQ ID NO:8); amino acid residue 518 to 523 (SEQ ID NO:9); aminoacid residue 658 to 663 (SEQ ID NO:10); and amino acid residue 190 to195 (SEQ ID NO:11). Antibodies generated from this immune response canbe isolated and purified as described herein. Methods for preparing andisolating polyclonal and monoclonal antibodies are well known in theart. See, for example, Current Protocols in Immunology, Cooligan, et al.(eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995;Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

[0162] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a zdint1 polypeptide or a fragment thereof.The immunogenicity of a zdint1 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of zdint1 or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0163] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

[0164] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to zdint1 proteinor peptide, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledzdint1 protein or peptide). Genes encoding polypeptides having potentialzdint1 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the zdint1sequences disclosed herein to identify proteins which bind to zdint1.These “binding proteins” which interact with zdint1 polypeptides can beused for tagging cells; for isolating homolog polypeptides by affinitypurification; they can be directly or indirectly conjugated to drugs,toxins, radionuclides and the like. These binding proteins can also beused in analytical methods such as for screening expression librariesand neutralizing activity. The binding proteins can also be used fordiagnostic assays for determining circulating levels of polypeptides;for detecting or quantitating soluble polypeptides as marker ofunderlying pathology or disease. These binding proteins can also act aszdint1 “antagonists” to block zdint1 binding and signal transduction invitro and in vivo. These anti-zdint1 binding proteins would be usefulfor inhibiting, for example, platelet aggregation, apoptosis,neurogenesis, myogenesis, tumor formation, and cell-cell interactions ingeneral.

[0165] Antibodies are determined to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related polypeptide molecules. First,antibodies herein specifically bind if they bind to a zdint1polypeptide, peptide or epitope with a binding affinity (Ka) of 10⁶ M⁻¹or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ orgreater, and most preferably 10⁹ M⁻¹ or greater. The binding affinity ofan antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad.Sci. 51: 660-672, 1949).

[0166] Second, antibodies are determined to specifically bind if they donot significantly cross-react with related polypeptides. Antibodies donot significantly cross-react with related polypeptide molecules, forexample, if they detect zdint1 but not known related polypeptides usinga standard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are orthologs, proteins from the same speciesthat are members of a protein family, zdint1 polypeptides, and non-humanzdint1. Moreover, antibodies may be “screened against” known relatedpolypeptides to isolate a population that specifically binds to theinventive polypeptides. For example, antibodies raised to zdint1 areadsorbed to related polypeptides adhered to insoluble matrix; antibodiesspecific to zdint1 will flow through the matrix under the proper bufferconditions. Such screening allows isolation of polyclonal and monoclonalantibodies non-crossreactive to closely related polypeptides(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995). Screening and isolation of specific antibodies iswell known in the art. See, Fundamental Immunology, Paul (eds.), RavenPress, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; MonoclonalAntibodies: Principles and Practice, Goding, J. W. (eds.), AcademicPress Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.

[0167] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to zdint1 proteinsor peptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zdint1protein or polypeptide.

[0168] Antibodies to zdint1 may be used for tagging cells that expresszdint1; for isolating zdint1 by affinity purification; for diagnosticassays for determining circulating levels of zdint1 polypeptides; fordetecting or quantitating soluble zdint1 as marker of underlyingpathology or disease; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzdint1 in vitro and in vivo. Suitable direct tags or labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmarkers, chemiluminescent markers, magnetic particles and the like;indirect tags or labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies herein mayalso be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zdint1or fragments thereof may be used in vitro to detect denatured zdint1 orfragments thereof in assays, for example, Western Blots or other assaysknown in the art.

[0169] The present invention also provides polypeptide fragments orpeptides comprising an epitope-bearing portion of a zacrp2 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat. Acad. Sci. USA81:3998, 1983).

[0170] In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660, 1983).Accordingly, antigenic epitope-bearing peptides and polypeptides of thepresent invention are useful to raise antibodies that bind with thepolypeptides described herein.

[0171] Antigenic epitope-bearing peptides and polypeptides preferablycontain at least four to ten amino acids, at least ten to fifteen aminoacids, or about 15 to about 30 amino acids of SEQ ID NO:2. Suchepitope-bearing peptides and polypeptides can be produced by fragmentinga zacrp2 polypeptide, or by chemical peptide synthesis, as describedherein. Moreover, epitopes can be selected by phage display of randompeptide libraries (see, for example, Lane and Stephen, Curr. Opin.Immunol. 5:268, 1993, and Cortese et al., Curr. Opin. Biotechnol. 7:616,1996). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-16 (The Humana Press, Inc.1992), Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997). Polypeptides, or fragmentsthereof, of the present invention comprising sequences of amino acidsfrom, for example, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:8, SEQ ID NO:10,or SEQ ID NO:11 are epitope bearing.

[0172] Bioactive conjugates:

[0173] Antibodies or polypeptides herein can also be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (integrin orantigen, respectively, for instance). More specifically, zdint1polypeptides or anti-zdint1 antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that expressthe anti-complementary molecule.

[0174] Suitable detectable molecules may be directly or indirectlyattached to the polypeptide or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Polypeptides or antibodies may also be conjugated tocytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the polypeptide or antibodyportion. For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

[0175] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a ligand binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

[0176] In another embodiment, zdint1-cytokine fusion proteins orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, brain, heart, spinal cord andskeletal muscle malignancies), if the zdint1 polypeptide or anti-zdint1antibody targets hyperproliferative brain, heart, spinal cord, orskeletal muscle cells. (See, generally, Hornick et al., Blood89:4437-47, 1997). They described fusion proteins that enable targetingof a cytokine to a desired site of action, thereby providing an elevatedlocal concentration of cytokine. Suitable zdint1 polypeptides oranti-zdint1 antibodies target an undesirable cell or tissue (i.e., atumor or a leukemia), and the fused cytokine mediated improved targetcell lysis by effector cells. Suitable cytokines for this purposeinclude interleukin 2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF), for instance.

[0177] In yet another embodiment, if the zdint1 polypeptide oranti-zdint1 antibody targets vascular cells or tissues, such polypeptideor antibody may be conjugated with a radionuclide, and particularly witha beta-emitting radionuclide, to reduce restenosis. Such therapeuticapproach poses less danger to clinicians who administer the radioactivetherapy. For instance, iridium-192 impregnated ribbons placed intostented vessels of patients until the required radiation dose wasdelivered showed decreased tissue growth in the vessel and greaterluminal diameter than the control group, which received placebo ribbons.Further, revascularisation and stent thrombosis were significantly lowerin the treatment group. Similar results are predicted with targeting ofa bioactive conjugate containing a radionuclide, as described herein.

[0178] The bioactive polypeptide or antibody conjugates described hereincan be delivered intravenously, intraarterially or intraductally, or maybe introduced locally at the intended site of action.

[0179] Uses of Polynucleotide/Polypeptide:

[0180] Molecules of the present invention can be used to identify andisolate receptors and integrins involved in cell-cell interactions. Forexample, proteins and peptides of the present invention can beimmobilized on a column and membrane preparations run over the column(Immobilized Affinity Ligand Techniques, Hermanson et al., eds.,Academic Press, San Diego, Calif., 1992, pp.195-202). Polypeptides andpeptides which bind to the zdint1 polypeptides, peptides, and variantsfo the present invention can then be eluted and characterized usingmethods known in the art. Proteins and peptides can also be radiolabeled(Methods in Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinitylabeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedanet al., Biochem. Pharmacol. 33:1167-80, 1984) and specific cell-surfaceproteins can be identified.

[0181] The molecules of the present invention will be useful in repairand remodeling after an ischemic event, and/or inhibiting plateletaggregation. The polypeptides, nucleic acid and/or antibodies of thepresent invention can be used in treatment of disorders associated withinfarct in brain or heart tissue, and/or platelet aggregation. Themolecules of the present invention can be used to modulate proteolysis,apoptosis, neurogenesis, myogenesis, connective tissue disorders,arthritis, chondrogenesis, cell adhesion, cell fusion, and signaling orto treat or prevent development of pathological conditions in suchdiverse tissue as heart, brain, spinal cord and skeletal muscle. Inparticular, certain diseases may be amenable to such diagnosis,treatment or prevention. The molecules of the present invention can beused to modulate inhibition and proliferation of neurons and myocytes inheart, brain, spinal cord and skeletal muscle tissues. Disorders whichmay be amenable to diagnosis, treatment or prevention with zdint1polypeptides include, for example, Alzheimers's Disease, tumorformation, Multiple Sclerosis, Congestive Heart Failure, IschemicReperfusion or infarct, and degenerative diseases.

[0182] The zdint1 molecules of the present invention may be particularlyuseful in the treatment of intimal hyperplasia or restenosis due toacute vascular injury. Acute vascular injuries are those which occurrapidly (i.e. over days to months), in contrast to chronic vascularinjuries (e.g. atherosclerosis) which develop over a lifetime. Acutevascular injuries often result from surgical procedures such as vascularreconstruction, wherein the techniques of angioplasty, endarterectomy,atherectomy, vascular graft emplacement or the like are employed.Hyperplasia may also occur as a delayed response in response to, e.g.,graft emplacement or organ transplantation. The dose of zdint1 in thetreatment for restenosis will vary with each patient but will generallybe in the range of those suggested above.

[0183] Advances in the treatment of coronary vascular disease includethe use of mechanical interventions to either remove or displaceoffending plaque material in order to re-establish adequate blood flowthrough the coronary arteries. Despite the use of multiple forms ofmechanical interventions, including balloon angioplasty, reductionatherectomy, placement of vascular stents, laser therapy, or rotoblator,the effectiveness of these techniques remains limited by anapproximately 40% restenosis rate within 6 months after treatment.

[0184] Restenosis is thought to result from a complex interaction ofbiological processes including platelet deposition and thrombusformation, release of chemotactic and mitogenic factors, and themigration and proliferation of vascular smooth muscle cells into theintima of the dilated arterial segment.

[0185] The inhibition of platelet accumulation at sites of mechanicalinjury can limit the rate of restenosis in human subjects. Therapeuticuse of a monoclonal antibody to platelet GpIIb/IIIa is able to limit thelevel of restenosis in human subjects (Califf et al., N. Engl. J. Med.,330: 956-961 (1994)). The antibody is able to bind to the GpIIb/IIIareceptor on the surfaces of platelets and thereby inhibit plateletaccumulation. This data suggests that inhibition of plateletaccumulation at the site of mechanical injury in human coronary arteriesis beneficial for the ultimate healing response that occurs.

[0186] Gene therapy:

[0187] Polynucleotides encoding zdint1 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzdint1 activity. If a mammal has a mutated or absent zdint1 gene, thezdint1 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zdint1 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0188] In another embodiment, a zdint1 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Feigner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0189] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0190] Antisense methodology can be used to inhibit zdint1 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zdint1-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:1) aredesigned to bind to zdint1-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof zdint1 polypeptide-encoding genes in cell culture or in a subject.

[0191] The present invention also provides reagents which will find usein diagnostic applications. For example, the zdint1 gene, a probecomprising zdint1 DNA or RNA or a subsequence thereof can be used todetermine if the zdint1 gene is present on chromosome 2q33 or if amutation has occurred. Detectable chromosomal aberrations at the zdint1gene locus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by employing molecular genetic techniques, suchas restriction fragment length polymorphism (RFLP) analysis, shorttandem repeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art (Sambrook et al., ibid.;Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).

[0192] Transgenic mice, engineered to express the zdint1 gene, orfragments thereof, and mice that exhibit a complete absence of zdint1gene function, referred to as “knockout mice” (Snouwaert et al., Science257:1083, 1992), can also be generated (Lowell et al., Nature366:740-42, 1993) by one skilled in the art. These mice can be employedto study the zdint1 gene, gene fragments, and the protein encodedthereby in an in vivo system.

[0193] Chromosomal Localization:

[0194] Radiation hybrid mapping is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-50, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Radiationhybrid mapping panels are commercially available which cover the entirehuman genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RHPanel (Research Genetics, Inc., Huntsville, Ala.). These panels enablerapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers. The precise knowledge of agene's position can be useful for a number of purposes, including: 1)determining if a sequence is part of an existing contig and obtainingadditional surrounding genetic sequences in various forms, such as YACs,BACs or cDNA clones; 2) providing a possible candidate gene for aninheritable disease which shows linkage to the same chromosomal region;and 3) cross-referencing model organisms, such as mouse, which may aidin determining what function a particular gene might have.

[0195] Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

[0196] For pharmaceutical use, the proteins of the present invention canbe administered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as powders, ointments,drops or transdermal patch) bucally, or as a pulmonary or nasalinhalant. Intravenous administration will be by bolus injection orinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include a zdint1 protein, alone, or inconjunction with a dimeric partner, in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water or the like. Formulations may further include one ormore excipients, preservatives, solubilizers, buffering agents, albuminto prevent protein loss on vial surfaces, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington:The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in therange of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20mg/kg per day, with the exact dose determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The proteins may beadministered for acute treatment, over one week or less, often over aperiod of one to three days or may be used in chronic treatment, overseveral months or years. In general, a therapeutically effective amountof zdint1 is an amount sufficient to produce a clinically significantchange in extracellular matrix remodeling, scar tissue formation, tumorsuppression, platelet aggregation, apoptosis, myogenesis, neurogenesis,electrical coupling, blood flow and/or cell proliferation in brain,heart, spinal cord, and skeletal muscle.

[0197] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Extension of EST Sequence

[0198] The novel zdint1 polypeptide-encoding polynucleotides of thepresent invention were initially identified by querying an EST database.This query identified an expressed sequence tag (EST) to nucleotide 1097to nucleotide 1415 of SEQ ID NO:1. A cDNA clone, corresponding to thisEST was obtained and the deduced amino acid sequence was determined tobe incomplete. Primers ZC17,991 (SEQ ID NO:4) and ZC17,992 (SEQ ID NO:5)were used to screen an arrayed fetal brain cDNA plasmid library toidentify clones of zdint1. Thermocycler conditions were as follows: onecycle at 94° C. for 1 minute 30 seconds; followed by thirty cycles at94° C. for 10 seconds, 64° C. for 20 seconds, 72° C. for 30 seconds,followed by one cycle at 72° C. for 5 minutes, followed by a 4° C. hold.A sample of the reaction contents was electrophoresed on a 4% agarosegel to identify positive pools. These pools were screened by polymerasechain reaction with ZC17,992 (SEQ ID NO:5) and the vector primerZC13,006 (SEQ ID NO:6). Thermocycler conditions were as follows: onecycle at 94° C. for 1 minute 30 seconds; followed by five cycles at 94°C. for 10 seconds, 68° C. for 2 minutes, followed by twenty-five cyclesat 94° C. for 10 seconds, 62° C. for 20 seconds, 72° C. for 2 minutes,followed by one cycle at 72° C. for 10 minutes, followed by a 4° C.hold. A sample of the reaction contents was electrophoresed on a 1%agarose gel and a band of ˜1.5 kb was further electrophoresed on a 1%preparative gel and the resulting band gel purified using commerciallyavailable gel purification reagents and protocol (QIAEX II GelExtraction Kit; Qiagen, Inc., Santa Clarita, Calif.). This fragment wassequenced and was determined to extend the amino acid sequence of zdint1in the 5′ direction.

Example 2 Tissue Distribution

[0199] Analysis of tissue distribution was performed by the Northernblotting technique using Human Multiple Tissue and Master Dot Blots fromClontech (Palo Alto, Calif.), and a human vascular tissue blot preparedin-house. The human vascular blot was prepared from the following celllines: Human Umbilical Vein Endothelial Cells (Cascade Biologics, Inc.,Portland, Oreg.), Human Pulmonary Artery Endothelial Cells (CascadeBiologics, Inc., Portland, Oreg.), Human Aortic Endothelail Cells,(Cascade Biologics, Inc., Portland, Oreg.), Aortic Smooth Muscle Cells(Clonetics, San Diego, Calif.), Human Intestinal Smooth Muscle Cells(American Type Culture Collectio, Manasas, Va.), Normal Human LungFibroblast, Clonetics, San Diego, Calif.) and Normal Human DermalFibroblast-Neonatal, Clonetics, San Diego, Calif.). Messenger RNA wasextracted and blots prepared by methods known in the art. The probe wasobtained by restriction digest of the original cDNA clone with arestriction endonuclease, PstI. The reaction mixture was electrophoresedon a preparative agarose gel and two bands, corresponding to a 239 basepair fragment and a 223 base pair fragment from the cDNA clone, were gelpurified using commercially available gel purification reagents andprotocol from Qiagen, Inc. A probe was made by pooling the purified DNAfrom both bands and was random prime labeled with ³²P using acommercially available kit (Rediprime DNA labeling system; AmershamCorp., Arlington Heights, Ill.). The probe was then purified using aNUCTRAP push column (Stratagene Cloning Systems, La Jolla, Calif.).EXPRESSHYB (Clontech) solution was used for pre-hybridization andhybridization. The hybridization solution consisted of 8 ml EXPRESSHYB,80 μl Sheared Salmon Sperm DNA (10 mg/ml, 5 Prime-3 Prime, Boulder,Colo.), 48 p1 Human Cot-1 DNA (1 mg/ml, Gibco BRL), and 57 μl labeledprobe (2.3×10⁻⁵ CPM/μl). Hybridization took place overnight at 50° C.,and the blots were then washed in 2× SSC and 0.1% SDS at ambient roomtemperature, then 2× SSC and 0.1% SDS at 60° C., followed by 0.1× SSCand 0.1% SDS at 60° C. The blots were exposed overnight and developed.Strong signals of three transcript sizes, approximately 3.0 kb, 4.4 kb,and 7.5 kb, were observed in heart on the multiple tissue Northernblots. Faint signals of the same transcript sizes were observed in brainand spinal cord. An fainter signal of the three transcript sizes wasobserved in skeletal muscle. The Master Dot Blot showed strong signalsin brain, heart, fetal brain, and fetal heart. For the human vascularblot, a strong signal at 3-3.5 kb in human aortic endothelial cells andweaker signals in aortic smooth muscle cells and normal human lungfibroblast cells was observed.

Example 3 Protein Purification

[0200] Purification conditions for zdint1 with N- and C-terminal EEtags:

[0201]E. coli, Pichia, CHO and BHK cells are transfected with expressionvectors containing the DNA sequence of SEQ ID NO:1, or a portionthereof, operably linked to a polynucleotide encoding a Glu-Glu tag.Zdintl protein is expressed in conditioned media of E. coli, Pichiamethanolica, and or chinese hamster ovary (CHO) and baby hamster kidney(BHK) cells. For zdint1 expressed in E. coli and Pichia, the media isnot concentrated prior to purification. Unless otherwise noted, alloperations are carried out at 4° C. A total of 25 liters of conditionedmedium from BHK cells is sequentially sterile filtered through a 4 inch,0.2 mM Millipore (Bedford, Mass.) OptiCap capsule filter and a 0.2 mMGelman (Ann Arbor, Mich.) Supercap 50. The material is then concentratedto about 1.3 liters using a Millipore ProFlux A30 tangential flowconcentrator fitted with a 3000 kDa cutoff Amicon (Bedford, Mass.) S10Y3membrane. The concentrated material is again sterile-filtered with theGelman filter, as described above. A mixture of protease inhibitors isadded to the concentrated conditioned medium to final concentrations of2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.Louis, Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis,Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). A 50.0 ml sample of anti-EE Sepharose, preparedas described below, is added and the mixture gently agitated on aWheaton (Millville, N.J.) roller culture apparatus for 18.0 h at 4° C.

[0202] The mixture is then poured into a 5.0×20.0 cm Econo-Column(Bio-Rad, Laboratories, Hercules, Calif.), and the gel is washed with 30column volumes of phosphate buffered saline (PBS). The unretainedflow-through fraction is discarded. Once the absorbance of the effluentat 280 nM is less than 0.05, flow through the column is reduced to zero,and the anti-EE Sepharose gel is washed with 2.0 column volumes of PBScontaining 0.2 mg/ml of EE peptide (AnaSpec, San Jose, Calif.). Thepeptide that is used has the sequence GluTyrMetProValAsp. After 1.0 h at4° C., flow is resumed and the eluted protein collected. This fractionis referred to as the peptide elution. The anti-EE Sepharose gel is thenwashed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycinewash is collected separately. The pH of the glycine-eluted fraction isadjusted to 7.0 by the addition of a small volume of 10× PBS and storedat 4° C. for future analysis, if needed.

[0203] The peptide elution is concentrated to 5.0 ml using a 15,000molecular weight cutoff membrane concentrator (Millipore, Bedford,Mass.), according to the manufacturer's instructions. The concentratedpeptide elution is then separated from free peptide by chromatography ona 1.5×50 cm Sephadex G-50 (Pharmacia, Piscataway, N.J.) columnequilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad SprintHPLC (PerSeptive BioSystems, Framingham, Mass.). Two-ml fractions arecollected and the absorbance at 280 nM monitored. The first peak ofmaterial absorbing at 280 nM and eluting near the void volume of thecolumn is collected. This fraction is pure zdint1 NEE or zdint1 CEE. Thepure material is concentrated as described above, analyzed by SDS-PAGEand Western blotting with anti-EE antibodies, aliquoted, and stored at−80° C. according to standard procedures.

[0204] Preparation of Anti-EE Sepharose:

[0205] A 100 ml bed volume of protein G-Sepharose (Pharmacia,Piscataway, N.J.) is washed 3 times with 100 ml of PBS containing 0.02%sodium azide using a 500 ml Nalgene 0.45 micron filter unit. The gel iswashed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma,St. Louis, Mo.). and an equal volume of EE antibody solution containing900 mg of antibody is added. After an overnight incubation at 4° C.,unbound antibody is removed by washing the resin with 5 volumes of 200mM TEA as described above. The resin is resuspended in 2 volumes of TEA,transferred to a suitable container, and dimethylpimilimidate-2HCl(Pierce, Rockford, Ill.), dissolved in TEA, is added to a finalconcentration of 36 mg/ml of gel. The gel is rocked at room temperaturefor 45 min and the liquid is removed using the filter unit as describedabove. Nonspecific sites on the gel are then blocked by incubating for10 min at room temperature with 5 volumes of 20 mM ethanolamine in 200mM TEA. The gel is then washed with 5 volumes of PBS containing 0.02%sodium azide and stored in this solution at 4° C.

[0206] Purification of Untagged zdint1

[0207]E. coli, Pichia, CHO and BHK cells are transfected with expressionvectors containing the DNA sequence of SEQ ID NO:1, or a portionthereof. The procedure described below is used for protein expressed inconditioned medium of E. coli, Pichia methanolica, and Chinese hamsterovary (CHO) and baby hamster kidney (BHK) cells. For zdint1 expressed inE. coli and Pichia, however, the medium is not be concentrated prior topurification. Unless otherwise noted, all operations are carried out at4° C. A total of 25 liters of conditioned medium from BHK cells issequentially sterile filtered through a 4 inch, 0.2 mM Millipore(Bedford, Mass.) OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor,Mich.) Supercap 50. The material is then be concentrated to about 1.3liters using a Millipore ProFlux A30 tangential flow concentrator fittedwith a 3000 kDa cutoff Amicon (Bedford, Mass.) S1OY3 membrane. Theconcentrated material is again be sterile-filtered with the Gelmanfilter as described above. A mixture of protease inhibitors is added tothe concentrated conditioned medium to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim).

[0208] The procedures outlined below are adaptations of those used topurify metalloprotease/disintegrins from Crotalus viridus and Crotalusatrox venom (Liu et al., Toxicol. 33: 1289-1298, 1995; Shimokawa et al.,Arch Biochem Biophys 343: 35-43, 1997). A combination of proceduresincluding, but not limited to, anion and cation exchange chromatography,size exclusion, and affinity chromography is used to purify untaggedzdint1.

[0209] Concentrated conditioned medium is diluted 1/10 in line with 10mM borate buffer, pH 9.0, 0.1 M NaCl, and 2.0 mM CaCl₂ using the BioCadSprint HPLC (PerSeptive BioSystems, Framingham, Mass.). The material ispumped onto a 3.5×20 cm Poros HQ (PerSeptive BioSystems, Framingham,Mass.) column at 5 ml/min. The column is washed with loading buffer, andwhen the absorbance of the effluent is less than 0.05, the column isdeveloped with a linear gradient of NaCl from 0.1 M to 1.0 M NaCl.Fractions containing zdint1 are identified by SDS-PAGE and Westernblotting with anti-zdint1 peptide antibodies. zdint1-containingfractions are pooled together, and concentrated using an Amicon stirredcell concentrator fitted with a YM-10 membrane. The Poros HQ pool isthen chromatographed on a Sephadex G-75 column equilibrated in 10 mMsodium phosphate, pH 7.0. Fractions containing zdint1 are identified andpooled together, as described above, and applied to a 1.0×5 cm Poros HAhydroxyapatite column at 1.0 ml/min using the BioCad Sprint HPLC. Thecolumn is washed with loading buffer and developed with a lineargradient from 10 mM to 500 mM sodium phosphate. Fractions contained purezdint1 are identified by SDS-PAGE and Western blotting, as describedabove. The purified material is aliquoted and stored as described above.

Example 4 Chromosomal Assignment and Placement of Zdint1

[0210] Zdint1 was mapped to chromosome 2 using the commerciallyavailable version of the “Stanford G3 Radiation Hybrid Mapping Panel”(Research Genetics, Inc., Huntsville, Ala.). The “Stanford G3 RH Panel”contains PCRable DNAs from each of 83 radiation hybrid clones of thewhole human genome, plus two control DNAs (the RM donor and the A3recipient). A publicly available WWW server(http://shgc-www.stanford.edu) allows chromosomal localization ofmarkers.

[0211] For the mapping of zdint1 with the “Stanford G3 RH Panel”, 20 μlreactions were set up in a PCRable 96-well microtiter plate (Stratagene,La Jolla, CA) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 85 PCR reactions consisted of 2 μl 10 ×KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto,Calif.), 1.6 pl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC20,843 (SEQ ID NO:12), 1 μl antisenseprimer, ZC20,844 (SEQ ID NO:13), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and distilled water for a total volume of 20 μl. Thereactions were overlaid with an equal amount of mineral oil and sealed.The PCR cycler conditions were as follows: an initial 1 cycle 5 minutedenaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94°C., 45 seconds annealing at 66° C. and 1 minute and 15 seconds extensionat 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.The reactions were separated by electrophoresis on a 2% agarose gel(Life Technologies, Gaithersburg, Md.).

[0212] The results showed linkage of Zdint1 to the framework markerSHGC-56733 with a LOD score of >12 and at a distance of 0 cR_(—)10000from the marker. The use of surrounding markers positions Zdint1 in the2q33 region on the integrated LDB chromosome 2 map (The Genetic LocationDatabase, University of Southhampton, WWW server:http://cedar.genetics.soton.ac.uk/public html/).

Example 5 Synthesis of Peptides

[0213] Zdint1-1, a peptide corresponding to amino acid residue 437 (Cys)to amino acid residue 450 (Cys) of SEQ ID NO:2, is synthesized by solidphase peptide synthesis using a model 431A Peptide Synthesizer (AppliedBiosystems/Perkin Elmer, Foster City, Calif.). Fmoc-Glutamine resin(0.63 mmol/g; Advanced Chemtech, Louisville, Ky.) is used as the initialsupport resin. 1 mmol amino acid cartridges (Anaspec, Inc. San Jose,Calif.) are used for synthesis. A mixture of 2(1-Hbenzotriazol-y-yl1,1,3,3-tetrahmethylhyluronium hexafluorophosphate (HBTU),1-hydroxybenzotriazol (HOBt), 2 m N,N-Diisolpropylethylamine,N-Methylpyrrolidone, Dichloromethane (all from Applied Biosystems/PerkinElmer) and piperidine (Aldrich Chemical Co., St. Louis, Mo.), are usedfor synthesis reagents.

[0214] The Peptide Companion software (Peptides International,Louisville, Ky.) is used to predict the aggregation potential anddifficulty level for synthesis for the zdint-1 peptide. Synthesis isperformed using single coupling programs, according to themanufacturer's specifications.

[0215] The peptide is cleaved from the solid phase following standardTFA cleavage procedure (according to Peptide Cleavage manual, AppliedBiosystems/Perkin Elmer). Purification of the peptide is done by RP-HPLCusing a C18, 10 μm semi-peparative column (Vydac, Hesperial, Calif.).Eluted fractions from the column are collected and analyzed for correctmass and purity by electrospray mass spectrometry. Pools of the elutedmaterial are collected. If pure, the pools are combined, frozen andlyophilized.

Example 6 Anticoagulant Activity of zdint1

[0216] The ability of the zdint1 protein to inhibit clotting is measuredin a one-stage clotting assay using wild-type zdint1 as a control.Recombinant proteins are prepared essentially as described above fromcells cultured in media containing 5 mg/ml vitamin K. Varying amounts ofthe zdint1 or recombinant wild-type zdint1 are diluted in 50 mM Tris pH7.5, 0.1% BSA to 100 ml. The mixtures are incubated with 100 ml ofzdint1-deficient plasma and 200 ml of thromboplastin C (Dade, Miami,Fla.; contains rabbit brain thromboplastin and 11.8 mM Ca⁺⁺). Theclotting assay is performed in an automatic coagulation timer (MLAElectra 800, Medical Laboratory Automation Inc., Pleasantville, N.Y.),and clotting times are converted to units of zdint1 activity using astandard curve constructed with 1:5 to 1:640 dilutions of normal pooledhuman plasma (assumed to contain one unit per ml zdint1 activity;prepared by pooling citrated serum from healthy donors).

[0217] Zdint1 activity is seen as a reduction in clotting time overcontrol samples.

Example 7 Inhibition of Platelet Accumulation with zdint1

[0218] Zdint1 is analyzed for its ability to inhibit plateletaccumulation at sites of arterial thrombosis due to mechanical injury innon-human primates. A model of aortic endarterectomy is utilized inbaboons, essentially as described by Lumsden et al. (Blood 81: 1762-1770removed, inverted and scraped to remove the intima of the artery andapproximately 50% of the media. The artery is reverted back to itscorrect orientation, cannulated on both ends and placed into anextracorporeal shunt in a baboon, thereby exposing the mechanicallyinjured artery to baboon blood via the shunt. Just prior to opening ofthe shunt to the circulating blood, ¹¹¹In-labeled autologous plateletsare injected intravenously into the animal. The level of plateletaccumulation at the site of the injured artery is determined byreal-time gamma camera imaging.

[0219] Evaluation of zdint1 for inhibition of platelet accumulation isdone using bolus injections of zdint1 or saline control and are givenjust prior to the opening of the shunt. The injured arteries aremeasured continuously for 60 minutes.

[0220] Zdint1 activity is seen as an inhibition of plateletaccumulation.

[0221] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

What is claimed is:
 1. An isolated polypeptide molecule comprising acontiguous sequence of 14 amino acids of SEQ ID NO:2.
 2. An isolatedpolypeptide molecule according to claim 1, wherein the polypeptidemolecule comprises residues 437 to 450 of SEQ ID NO:2.
 3. An isolatedpolypeptide molecule according to claim 1 wherein the polypeptidemolecule is between 82 and 232 amino acids in length.
 4. An isolatedpolypeptide molecule according to claim 3 wherein the polypeptidemolecule is residues 164 to 382 of SEQ ID NO:2.
 5. An isolatedpolypeptide molecule according to claim 3 wherein the polypeptidemolecule is residues 383 to 464 of SEQ ID NO:2.
 6. An isolatedpolypeptide molecule according to claim 3 wherein the polypeptidemolecule is residues 465 to 696 of SEQ ID NO:2.
 7. A isolatedpolypeptide molecule selected from the group consisting of: a) apolypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; b) apolypeptide molecule comprising residues 383 to 464 of SEQ ID NO:2; c) apolypeptide molecule comprising residues 465 to 696 of SEQ ID NO:2; d) apolypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; e) apolypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2; f) apolypeptide molecule comprising residues 164 to 696 of SEQ ID NO:2; g) apolypeptide molecule comprising residues 383 to 696 of SEQ ID NO:2; h) apolypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2; i) apolypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2; andj) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO:2.8. An isolated polynucleotide molecule encoding a polypeptide molecule,wherein the polypeptide molecule comprises a contiguous sequence of 14amino acids of SEQ ID NO:2.
 9. An isolated polynucleotide moleculeaccording to claim 8, wherein the polypeptide molecule comprisesresidues 437 to 450 of SEQ ID NO:2.
 10. An isolated nucleotide moleculeaccording to claim 8, wherein the polypeptide molecule is between 82 and232 amino acids in length.
 11. An isolated polynucleotide moleculeaccording to claim 10, wherein the polypeptide molecule is residues 164to 382 of SEQ ID NO:2.
 12. An isolated polynucleotide molecule accordingto claim 10, wherein the polypeptide molecule is residues 383 to 464 ofSEQ ID NO:2.
 13. An isolated polynucleotide molecule according to claim10, wherein the polypeptide molecule is residues 465 to 696 of SEQ IDNO:2.
 14. A isolated polynucleotide molecule encoding a polypeptidemolecule, wherein the polypeptide molecule is selected from the groupconsisting of: a) a polypeptide molecule comprising residues 164 to 382of SEQ ID NO:2; b) a polypeptide molecule comprising residues 383 to 464of SEQ ID NO:2; c) a polypeptide molecule comprising residues 465 to 696of SEQ ID NO:2; d) a polypeptide molecule comprising residues 438 to 449of SEQ ID NO:2; e) a polypeptide molecule comprising residues 164 to 464of SEQ ID NO:2; f) a polypeptide molecule comprising residues 164 to 696of SEQ ID NO:2; g) a polypeptide molecule comprising residues 383 to 696of SEQ ID NO:2; h) a polypeptide molecule comprising residues 164 to 449of SEQ ID NO:2; i) a polypeptide molecule comprising residues 438 to 696of SEQ ID NO:2; and j) a polypeptide molecule comprising residues 1 to696 of SEQ ID NO:2.
 15. An isolated polynucleotide encoding a fusionprotein having a first segment and a second segment, wherein the firstsegment comprises a first polypeptide encoding a polypeptide having aprotease domain and the second segment comprises a second polynucleotideencoding a polypeptide that has a contiguous sequence of 14 amino acidsbetween residues 383 and 464 of SEQ ID NO:2, and wherein the firstsegment is positioned amino-terminally to the second segment.
 16. Anisolated polynucleotide according to claim 15, wherein the proteasedomain is selected from the group consisting of; a) a protease domainthat is a member of the Disintegrin Proteases; and b) a protease domainthat is at least 80% identical to amino acid residues 164 to 382 of SEQID NO:2.
 17. An isolated polynucleotide molecule encoding a polypeptidemolecule wherein the polynucleotide molecule is selected from the groupconsisting of: a) a polynucleotide molecule that encodes a polypeptidemolecule that is at least 80% identical to residues 383 to 464 of SEQ IDNO:2; and b) a polynucleotide molecule that is complementary to a). 18.An isolated polynucleotide molecule according to claim 17 wherein thepolynucleotide molecule is selected from the group consisting of: a) apolynucleotide molecule that encodes a polypeptide molecule that is atleast 80% identical to residues 383 to 696 of SEQ ID NO:2; and b) apolynucleotide molecule that is complementary to a).
 19. An isolatedpolynucleotide molecule according to claim 17, wherein thepolynucleotide molecule is selected from the group consisting of: a) apolynucleotide molecule that encodes a polypeptide molecule that is atleast 80% identical to residues 1 to 696 of SEQ ID NO:2; and b) apolynucleotide molecule that is complementary to a).
 20. An expressionvector comprising the following operably linked elements: a) atranscription promoter; b) a DNA segment encoding the polypeptide ofclaim 1; and c) a transcription terminator.
 21. An expression vector ofclaim 20 wherein the DNA segment further encodes an affinity tag.
 22. Acultured cell into which has been introduced an expression vectoraccording to claim 21, wherein said cell expresses the polypeptideencoded by the DNA segment.
 23. A method of producing a polypeptidecomprising culturing a cell according to claim 22, whereby said cellexpresses the polypeptide encoded by the DNA segment; and recovering thepolypeptide.
 24. A method for modulating cell-cell interactions bycombining the polypeptide according to claim 1, with cells in vivo andin vitro.
 25. A method for modulating cell-cell interactions accordingto claim 24, whereby the cells are derived from tissues selected fromthe group consisting of: a) tissues from heart; b) tissues from brain;c) tissues from spinal cord; and d) tissues from skeletal muscle.
 26. Anisolated polypeptide molecule comprising a contiguous sequence of aminoacids, wherein the contiguous sequence of amino acids is selected fromthe group consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8; c) SEQ ID NO:9;d) SEQ ID NO:10; and e) SEQ ID NO:11.
 27. An isolated polynucleotidemolecule encoding the isolated polypeptide molecule of claim 26.